Compositions and methods of preparing Leptospira

ABSTRACT

The present invention includes compositions and methods of preparing flagellar-coiling protein 1 (Fcp1)-deficient Leptospira bacterium. In one aspect, the invention includes an isolated, flagellar-coiling protein 1 (Fcp1)-deficient Leptospira bacterium. Another aspect includes a composition comprising a flagellar-coiling protein 1 (Fcp1) deficient Leptospira bacterium. Yet another aspect includes a method of producing a motility-deficient Leptospira bacterium comprising inhibiting expression of a wild-type flagellar-coiling protein 1 (Fcp1) gene. Methods of stimulating an immune response and reducing or treating an infectious disease caused by one or more Leptospira bacteria in a subject in need thereof comprising administering a composition comprising an effective amount of flagellar-coiling protein 1 (Fcp1) deficient Leptospira bacteria to the subject are also included.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of, and claims priority to, U.S.patent application Ser. No. 15/124,633, filed Sep. 8, 2016, now U.S.Pat. No. 10,143,735, which is a 35 U.S.C. § 371 national phaseapplication from, and claims priority to, International Application No.PCT/US2015/019865, filed Mar. 11, 2015, and published under PCT Article21(2) in English, which is entitled priority under 35 U.S.C. § 119(e) toU.S. Provisional Application No. 61/951,734, filed Mar. 12, 2014, all ofwhich applications are incorporated herein by reference in theirentireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under AI0188752 andAI052473 awarded by National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Leptospirosis is a life-threatening disease, which can occur in adiverse range of epidemiological situations. The spirochetal agent is aunique, albeit genetically and antigenically diverse group of bacteriadivided into eight pathogenic Leptospira species and >200 serovars. Thedisease is considered the most widespread zoonosis in the world due tothe pathogen's ability to induce a carrier state in a wide range of wildand domestic animals. Leptospires are highly motile bacteria, which canpenetrate abraded skin and mucous membranes, causing a systemicinfection in a short period of time by crossing tissue barriers and byhaematogenous dissemination.

Spirochetes are one of the only phyla of bacteria that can be recognizedand identified based on their unique and distinct corkscrew or helicalmorphologies. Cells of Leptospira spp., which have single shortperiplasmic flagella (PFs) at each end without overlapping at the centerof the cell, exhibit a number of different shapes when moving. Duringtranslational motility, the anterior end is a spiral-shape and theposterior end is a hook-shaped, with translating cells rotating theirPFs in opposite directions. Viewed from the center of the cell towardsone of the cell ends, counterclockwise rotation results in thespiral-shaped end, and clockwise rotation results in the hook-shaped.They can readily reverse directions together with shape, and if the PFsare rotating in the same direction, the cells do not translate. Whenisolated in vitro and observed by negative-stain electron microscopy,PFs are extensively coiled in the form of a spring, and when the cellsare at rest, fixed or dead, they differ from most other spirochetes forthe presence of hook-shaped ends. Berg et al. (Berg et al., 1978, pp.285-295, Cambridge: Cambridge University Press) proposed that inLeptospira, the PFs are more rigid when compared to the cell cylinder.Furthermore, previous studies showed that mutants that form uncoiledPFs, or without PFs, still maintain their helical shape, but havestraight ends, which impairs their normal motility phenotype (Bromley etal., 1979, J Bacteriol 137, 1406-1412; Picardeau et al., 2001, MolMicrobiol 40, 189-199). Those results, taken together, indicate that thedirection of rotation of PFs, and its interaction with the helicallyshaped cell cylinder determines a different conformation of the cell'sends, allowing them to move efficiently in both viscous media and lowviscosity solvents.

PFs in spirochetes are known to be rather similar in structure andfunction to the flagella of other externally flagellated bacteria, aseach consists of a basal body-motor complex, and a flexible hook regionthat connects to a helical flagellar filament. However, flagellarfilaments of spirochete PFs are unique and complex, composed by multipleproteins, whereas in other swimming bacteria the Flagellin protein alonecomposes a thinner flagellar filament. A family of proteins named FlaBis consistently claimed to form the core of PF in spirochetes, and itshows sequence similarity with flagellin. However, spirochetes specieshave at least one additional set of proteins designated FlaA, whichsurrounds the inner core to form a sheath or partial sheath of T.pallidum, and B. hyodysenteriae PFs, but not in the sheath of B.burgdorferi PFs.

Although the function of the flagellar sheath is not clear, it has beensuggested that it increases the stiffness of PFs, and that this producesa greater swimming velocity. In a more recent study, random mutants withdisruption of both flaA genes in L. interrogans indicated that FlaAproteins have no involvement in the formation of the PFs sheath in thisspecies.

Although motility and hook-deficient mutants were previously described,linking genes to protein function in these bacteria has been difficultdue to the lack of tools for genetic manipulation of Leptospira.

Therefore, a need exists in the art for understanding the compositionand architecture of PFs, and the role that motility plays in leptospiralvirulence.

SUMMARY OF THE INVENTION

As described herein, the present invention includes methods andcompositions of preparing flagellar-coiling protein 1 (Fcp1)-deficientLeptospira bacterium and methods of stimulating an immune response andreducing or treating an infectious disease caused by one or moreLeptospira bacteria in a subject in need thereof.

One aspect of the invention includes an isolated, flagellar-coilingprotein 1 (Fcp1)-deficient Leptospira bacterium. In another aspect, theinvention includes a composition comprising a flagellar-coiling protein1 (Fcp1) deficient Leptospira bacterium. In yet another aspect, theinvention includes a vaccine comprising an effective amount of amotility deficient Leptospira bacteria. In still another aspect, theinvention includes a composition for stimulating an immune response in asubject in need thereof comprising an effective amount of a motilitydeficient Leptospira bacteria.

In one aspect, the invention includes a method of stimulating an immuneresponse in a subject in need thereof comprising administering acomposition comprising an effective amount of a motility deficientLeptospira bacteria to the subject. In another aspect, the inventionincludes a method for reducing or treating an infectious disease causedby one or more Leptospira bacteria in a subject in need thereofcomprising administering a composition comprising an effective amount ofa motility deficient Leptospira bacteria to the subject.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the Leptospira bacterium comprises asilenced or deleted Fcp1 gene or a mutant Fcp1 gene. In one embodiment,the mutant Fcp1 gene expresses a mutant protein incapable of functioningas wildtype Fcp1. In another embodiment, the Leptospira bacterium ismotility-deficient. In yet another embodiment, the Leptospira bacteriumhas attenuated bacterial virulence. In still another embodiment, theLeptospira bacterium is at least one of non-pathogenic, a livebacterium, and heat-inactivated.

In another embodiment, the Leptospira bacteria are at least one of livebacteria, heat-inactivated, and have attenuated bacterial virulence. Inyet another embodiment, the Leptospira bacteria is deficit in awild-type protein selected from the group consisting of flbB, flbD,flgA, flgB, flgC, flgD, flgG, flgH, flgI, flgM, flhA, flhB, flhF, flhX,fliA, fliE, fliF, fliG, fliG1, fliG3, fliH, fliI, fliJ, fliL, fliM,fliN, fliO, flip, fliQ, fliR, motA, motA1, motB, motB, flgE, flgJ, flgK,flgL, flhO, fliD, fliK, fcp1, fcp2, flaA1, flaA2, flaB1, flaB2, flaB3,flaB4, fliS, and any combination thereof.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the composition further comprises apharmaceutically acceptable carrier. In one embodiment, the compositionfurther comprises an adjuvant, such as an oil-in-water emulsion, asaponin, a cholesterol, a phospholipid, a CpG, a polysaccharide,variants thereof, and a combination thereof.

Another aspect of the invention includes a method of producing amotility-deficient Leptospira bacterium comprising inhibiting expressionof a wild-type flagellar-coiling protein 1 (Fcp1) gene.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, inhibiting expression of the wild-type Fcp1gene comprises deleting or silencing the wild-type Fcp1 gene in theLeptospira bacterium. In another embodiment, inhibiting expression ofthe wild-type Fcp1 gene comprises mutating the wild-type Fcp1 gene inthe Leptospira bacterium. In such an embodiment, the mutant Fcp1 geneexpresses a mutant Fcp1 protein incapable of functioning as wildtypeFcp1 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a panel of pictures showing motility assays using 0.5%agarose EMJH medium. Approximately 10⁵ leptospires were inoculated, andplates were incubated for 10 days at 30° C. Each square has 1 cm;

FIG. 1B is a panel of pictures showing the morphology of the clonesobserved by dark field microscopy using 100× oil objective and darkfield oil condenser;

FIG. 1C is a panel of scans showing the morphology of the cells observedby scanning electron microscopy;

FIG. 1D is a panel of scans showing the morphology of purifiedperiplasmic flagella (PF) observed by transmission electron microscopyusing 2% phosphotungstic acid (PTA) negative staining.

FIG. 2A is a gel showing the detection of Fcp1 expression. L.interrogans Fiocruz LV 2756 motile (lane 1), L. interrogans Fiocruz LV2756 motility-deficient (lane 2), L. interrogans Fiocruz LV 2756motility-deficient fcp1⁺ (lane 3), L. interrogans Fiocruz L1 130 WT(lane 4), L. interrogans Fiocruz L1 130 fcp1⁻ (lane 5), and L.interrogans Fiocruz L1 130 fcp1^(−/+) (lane 6). SDS/PAGE. Arrowsindicate the position of the proteins identified by mass spectrometry;

FIG. 2B is a western blot of purified PFs. Western Blot was probe withpolyclonal antibodies against Fcp1, and polyclonal antibodies againstFlaA1. Arrows indicate the position of the proteins identified byspecific polyclonal antibodies;

FIG. 3 diagrams the inactivation of the fcp1 gene. Schematicrepresentation of the genotype of L. interrogans Fiocruz L1 130 WT andL. interrogans Fiocruz L1 130 fcp1⁻, showing the allelic exchange of thefcp1 gene by Spc^(r) cassette. The highlighted area is indicating theregion of the fcp1 gene where the mutation occurred in the Fiocruz LV2756 motility-deficient, showing also the result regarding the proteinexpression;

FIG. 4A shows the cell morphology with a cryo-ET analysis of the FiocruzL1-130 WT;

FIG. 4B shows the cell morphology with a cryo-electron tomography (ET)analysis of the Fiocruz L1-130 fcp1⁻;

FIG. 4C shows the cell morphology with a cryo-ET analysis of the FiocruzL1-130 fcp1^(−/+);

FIG. 4D is a tomographic reconstruction showing one slice of FiocruzL1-130 WT. Flagellar filaments are indicated by arrays, and the picturesare from the averaged maps of PFs segments from each of the strains. Thediameter of the flagellar filament in Fiocruz L1-130 fcp1⁻ mutant is15.7 nm, in contrast to the diameter of 20.5 nm in wild-type organismsor complemented cells;

FIG. 4E is a tomographic reconstruction showing one slice of L1-130fcp1⁻ and L1-130 fcp1^(−/+). Flagellar filaments are indicated byarrays, and the pictures are from the averaged maps of PFs segments fromeach of the strains. The diameter of the flagellar filament in FiocruzL1-130 fcp1⁻ mutant is 15.7 nm, in contrast to the diameter of 20.5 nmin wild-type organisms or complemented cells;

FIG. 4F is a tomographic reconstruction showing one slice of L1-130fcp1^(−/+). Flagellar filaments are indicated by arrays, and thepictures are from the averaged maps of PFs segments from each of thestrains. The diameter of the flagellar filament in Fiocruz L1-130 fcp1⁻mutant is 15.7 nm, in contrast to the diameter of 20.5 nm in wild-typeorganisms or complemented cells;

FIG. 4G shows the surface renderings of 3-D reconstructions of FiocruzL1-130 fcp1⁻ with prominent structural features including the outermembrane (OM), cytoplasmic membrane (IM) and flagellar filament;

FIG. 4H shows the surface renderings of 3-D reconstructions of FiocruzL1-130 fcp1^(−/+), with prominent structural features including theouter membrane (OM), cytoplasmic membrane (IM) and flagellar filament;

FIG. 5A is a panel of scans showing an immuno-EM assay using purifiedPFsI preparation from Fiocruz L1-130 WT. The PF was labeled withantibodies against Fcp1 (α-Fcp1), FlaB1 (α-FlaB1), FlaA1 (α-FlaA1), andFlaA2 (α-FlaA2). Secondary antibody anti-rabbit conjugated with 5 nmgold nanoparticles was used to detect bound antibodies. PF werevisualized using 2% PTA negative staining;

FIG. 5B is a panel of scans showing an immuno-EM assay using purifiedPFsI preparation from Fiocruz L1-130 fcp1⁻. The PF was labeled withantibodies against Fcp1 (α-Fcp1), FlaB1 (α-FlaB1), FlaA1 (α-FlaA1), andFlaA2 (α-FlaA2). Secondary antibody anti-rabbit conjugated with 5 nmgold nanoparticles was used to detect bound antibodies. PF werevisualized using 2% PTA negative staining; and

FIG. 6A is a graph showing the dissemination of leptospires in tissuesfrom hamsters infected with the Fiocruz LV 2756 motile (white columns),and the Fiocruz LV 2756 motility-deficient (gray columns) determined byquantitative real time PCR. The analysis of the tissues was performedone day post-infection;

FIG. 6B is a graph showing the dissemination of leptospires four dayspost-infection with 10⁸ leptospires infected by intraperitoneal route;

FIG. 6C is a graph showing the dissemination of leptospires after 7 dayspost-infection with 10⁸ leptospires infected by conjunctival route.Bacterial load for each tissue was calculated based on the mean resultof two perfused hamsters. Each column in FIGS. 5A-5C represents the mean(logarithmic scale) of two independent experiments. Error bars in FIGS.5A-5C represent the standard deviation;

FIG. 7 is a graph showing the results of translocation assays and thepercent recovery of leptospires after inoculation of polarized MDCK cellmonolayers with Fiocruz L1-130 WT, Fiocruz L1-130 fcp1⁻, and FiocruzL1-130 fcp1^(−/+). Bacteria were inoculated in the upper chamber of MDCKcell monolayer transwell chambers. Translocating bacteria was quantifiedby counting bacteria in the lower chamber. Assays were performed at 2,4, 6, and 24 hours after addition of bacteria. The assays were performedin triplicate, and results are expressed as mean±SD.

FIG. 8 is a graph showing the kinetics of dissemination aftersubcutaneous inoculation with 10⁷ bacteria of L1-130 wild-type,attenuated vaccine and heat-killed vaccine in blood, kidney, liver andbrain between days 1 up to 21-days post infection.

FIG. 9A is an image of a western blot illustrating the profile of a poolof sera from hamsters 21 days post-vaccination with the live-attenuatedfcp1⁻ vaccine (before challenge).

FIG. 9B is an image of a western blot illustrating the profile of a poolof sera from hasmters 21 days post-vaccination with the heat-killed L.interrogans L1-130 vaccine (before challenge).

FIG. 10 is a survival curve of passive transfer with sera from hamstervaccinated with L. interrogans serovar Fiocruz L1-130 fcp1⁻ afterintraperitoneal heterologous challenge with 10⁷ bacteria of L.interrogans strain Manilae.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein may be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

As used herein, the articles “a” and “an” are used to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein when referring to a measurable value such as an amount, atemporal duration, and the like, the term “about” is meant to encompassvariations of ±20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.1%, 0.05%, or 0.01% of the specified value, as such variationsare appropriate to perform the disclosed methods. Unless otherwise clearfrom context, all numerical values provided herein are modified by theterm about.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies includeintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

By “attenuated” is meant the bacterium has a decreased virulence withrespect to a wild-type bacterium. In particular, a bacterium has anattenuated virulence of about 10, 20, 30, 40, 50, 60, 70, 80% or moredecrease in virulence as compared to a wild-type bacterium.

By “flagellar-coiling protein 1 gene” or “Fcp1 gene” is meant a nucleicacid molecule encoding a Fcp1 gene or fragment thereof. An exemplaryFcp1 gene sequence corresponds to a hypothetical protein coded by thegene LIC13166 found on chromosome 1 of a L. interrogans serovarCopenhageni strain 2756 in the GenBank Accession No. NC_005823 and isprovided in SEQ ID NO:1 and below:

  1 gtgagcatta tgaaggtgat gaaaagcata ttcattcttc tggccgtgct gggactcaac 61 ctgtctgttt tagctcagca aaacaatcag ggcggtaatc agcaagccaa cgaatccgta121 gaaaaaattg atgagctgtt aaaaggcgag ttggttcccg aagacgatga caaaaacctc181 acggaagagc agaagcgtcg taaaaaagca attcaggaac aagaagctct gtggaaaaac241 cctgatttta agggctatga taaaaatttc caagaactcc accaactctc caaagcattc301 gcgaacaaca aatttaggtt ggcattatcc aattaccaat cgggcgttaa cacgattctt361 aaaatgagag aagccataga acaataccgc aaagaagaag ctgaaaaaaa gcgtctcgat421 gaaaagtggt actggcaaaa agtagatcgt aaggcgagag aagaccgtgt cgtttctaga481 gacaaactag ttgccaaaca acaggcttta aattatttca ccaaggcgat caatcatttg541 gatgaaatca aaaacccaga cttgagagaa agaccggagt tcaaaagact tctttccgat601 acttacagat cttggatcct taccgaatac gatttacaaa atcttcctca gtgtatcccc661 attctcgagc tctatatcga gatcgatgaa aatgaaaagg aatatcctgc tcataagtat721 ctagcaagtt gttacgcttt cgaagaaaac atgatcaaaa agaatggtgg agcatccgaa781 gatcagatgt tcaaataccg ttataagaaa aacgttcacc ttttgagagc gactgaactg841 aagtatggaa aggattctcc cgaatacaaa cacatcgtta atcttgtaaa caaggacgaa901 gtgatttcgg ttagacctta a

As used herein, the term “fragment” as applied to a nucleic acid, isless than about 950 nucleotides in length or less than the whole Fcp1gene. In one embodiment, a fragment is between about 700 nucleotides toabout 900 nucleotides in length, preferably, between at least about 600nucleotides to about 950 nucleotides in length, more preferably, betweenabout at least about 500 nucleotides to about 1000 nucleotides inlength, even more preferably, between at least about 200 nucleotides toabout 700 nucleotides, yet even more preferably, between at least about100 nucleotides to about 950 nucleotides, and yet even more preferably,between at least about 50 nucleotides to about 1000 nucleotides inlength, and most preferably, the nucleic acid fragment will be greaterthan about 700 nucleotides in length.

By “flagellar-coiling protein 1 protein” or “Fcp1 protein” is meant aprotein or fragment thereof having at least about 85% amino acididentity to the hypothetical protein LIC13166 encoded by the geneLIC13166 found on chromosome 1 of a L. interrogans serovar Copenhagenistrain 2756 in the amino acid sequence of GenBank Accession No.YP_003074.1, or a fragment thereof, and having at least one Fcp1 proteinbiological activity. In one embodiment, a Fcp1 protein has at leastabout 85% amino acid sequence identity to SEQ ID NO:2 and the followingamino acid sequence:

  1 msimkvmksi fillavlgln lsvlaqqnnq ggnqqanesv ekidellkge lvpedddknl 61 teeqkrrkka iqeqealwkn pdfkgydknf qelhqlskaf annkfrlals nyqsgvntil121 kmreaieqyr keeaekkrld ekwywqkvdr karedrvvsr dklvakqqal nyftkainhl181 deiknpdlre rpefkrllsd tyrswiltey dlqnlpqcip ilelyieide nekeypahky241 lascyafeen mikknggase dqmfkyrykk nvhllratel kygkdspeyk hivnlvnkde301 visvrp

As applied to a protein, a “fragment” of Fcp1 protein is about 50 aminoacids in length. More preferably, the fragment of Fcp1 protein is about75 amino acids, even more preferably, at least about 100, yet morepreferably, at least about 125, even more preferably, at least about150, yet more preferably, at least about 200, even more preferably,about 225, and more preferably, at least about 250, and more preferably,at least about 300 amino acids in length amino acids in length.

By “Fcp1 deficient” is meant a bacterium that lacks wildtype Fcp1proteins or lacks the wildtype Fcp1 gene. For example, a Leptospirabacterium that has a Fcp1 gene that is silenced is Fcp1 deficient inwildtype Fcp1 protein. In another embodiment, a Leptospira bacteriumthat has a deleted Fcp1 gene is Fcp1 deficient in both the Fcp1 gene andFcp1 proteins. In another embodiment, a Leptospira bacterium that has amutated Fcp1 gene is Fcp1 deficient in both the wildtype Fcp1 gene andwildtype Fcp1 proteins.

By “heat-inactivated” refers to the process or method of heating apathogen, such as bacteria, to temperatures sufficiently high thatirreversible denaturation of proteins, such as membrane proteins,ribosomes, and nucleic acids, occurs. Heat-inactivated bacteria haveattenuated virulence and may be non-pathogenic.

As applied to the nucleic acid or protein, “homologous” as used hereinrefers to a sequence that has about 50% sequence identity. Morepreferably, the homologous sequence has about 75% sequence identity,even more preferably, has at least about 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% sequence identity.

By “host” or “host cell” is meant a cell, such as a mammalian cell, thatharbors a pathogen, such as a Leptospira bacterium. The pathogen caninfect the host cell is capable of infecting.

By “immune response” is meant the actions taken by a host to defenditself from pathogens or abnormalities. The immune response includesinnate (natural) immune responses and adaptive (acquired) immuneresponses. Innate responses are antigen non-specific. Adaptive immuneresponses are antigen specific. An immune response in an organismprovides protection to the organism against Leptospira bacterialinfections when compared with an otherwise identical subject to whichthe composition or cells were not administered or to the human prior tosuch administration.

By “infection” is meant a bacterial colonization of the host. Infectionof a host can occur by entry of the bacterium or bacteria through abreak in barrier epithelial surfaces, such as unhealed breaks in theskin, the eyes, or with the mucous membranes.

By “Leptospira bacterium” or “Leptospira bacteria” is meant a spirochetebacterium or bacteria. Leptospira bacteria are very thin, tightlycoiled, obligate aerobic spirochetes characterized by a unique flexuoustype of motility. Leptospira bacterium is a gram-negative spirochetewith internal flagella. The genus is divided into two species: thepathogenic leptospires L. interrogans and the free-living leptospire L.biflexa. Serotypes of L. interrogans are the agents of leptospirosis, azoonotic disease.

By “functional” or “functioning” is meant to have at least one Fcp1protein biological activity.

By “motility-deficient” is meant the inability of a Leptospira bacteriumto move or swim in a solution having a certain viscosity. Examples ofgenes that affect motility in Leptospira bacterium include, but are notlimited to, flbB, flbD, flgA, flgB, flgC, flgD, flgG, flgH, flgI, flgM,flhA, flhB, flhF, flhX, fliA, fliE, fliF, fliG, fliG1, fliG3, fliH,fliI, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, motA, motA1, motB,motB, flgE, flgJ, flgK, flgL, flhO, fliD, fliK, fcp1, fcp2, flaA1,flaA2, flaB1, flaB2, flaB3, flaB4, fliS, and any combination thereof.

By “mutant” or “mutated” is meant a change of the nucleotide sequence ina gene of a Leptospira bacterium, such as a flagellar-coiling protein 1(Fcp1) gene. The mutant or mutated gene can result in several differenttypes of change in sequences, such as altering the Fcp1 protein, orpreventing a gene from functioning properly or completely. Mutations canalso occur in nongenic regions of a gene, such that expression of a geneis altered, decreased, or not expressed.

By “pathogen” is meant an infectious agent, such as Leptospira bacteria,capable of causing infection, producing toxins, and/or causing diseasein a host.

By “silence” or “silenced” is meant that expression of the Fcp1 gene isprevented or decreased. Fcp1 gene silencing can occur via a geneknockdown, such as RNAi. When genes are knocked down, their expressionis decreased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90% or more.

By the term “vaccine” as used herein, is meant a composition, abacterium, a protein, or a nucleic acid of the invention, which servesto protect an animal against a Leptospira bacterial disease and/or totreat an animal already infected with Leptospira bacteria compared withan otherwise identical animal to which the vaccine is not administeredor compared with the animal prior to the administration of the vaccine.

By “virulence” is meant a degree of pathogenicity of a given pathogen orthe ability of an organism to cause disease in another organism.Virulence refers to an ability to invade a host organism, cause disease,evade an immune response, and produce toxins.

By “bacterial virulence” is meant a degree of pathogenicity of bacteria,such as Leptospira bacteria. Bacterial virulence includes causinginfection or disease in a host, producing agents that cause or enhancedisease in a host, producing agents that cause or enhance disease spreadto another host, and causing infection or disease in another host.

By “virulent” or “pathogenic” is meant a capability of a bacterium tocause a severe disease.

By “non-pathogenic” is meant an inability to cause disease.

By “wildtype” is meant a non-mutated version of a gene, allele,genotype, polypeptide, or phenotype, or a fragment of any of these. Itmay occur in nature or produced recombinantly.

By “wildtype Fcp1” is meant a nucleic acid molecule, a gene or a proteinthat contains a native Fcp1 nucleic acid sequence, gene, or amino acidsequence.

By “infectious disease” is meant a disease or condition in a subjectcaused by a pathogen that is capable of being transmitted orcommunicated to a non-infected subject. Non-limiting examples ofinfectious diseases include bacterial infections, viral infections,fungal infections, and the like.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “portion” of a polynucleotide means at least at least about twentysequential nucleotide residues of the polynucleotide. It is understoodthat a portion of a polynucleotide may include every nucleotide residueof the polynucleotide.

By “effective amount” is meant the amount required to reduce or improveat least one symptom of a disorder, condition or disease relative to anuntreated patient. The effective amount used for therapeutic treatmentof a condition or disease or stimulating an immune response, variesdepending upon the manner of the specific disorder, condition ordisease, extent of the disorder, condition or disease, andadministration of the cells, as well as the age, body weight, andgeneral health of the subject.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

The term “isolated” refers to a material or an organism, such asbacteria, that is free to varying degrees from components or otherorganisms which normally accompany it as found in its native state.Isolated denotes a degree of separation from an original source orsurroundings. An isolated bacterium is sufficiently free of otherbacteria such that any contaminants do not materially affect growth,pathogencity, infection, etc. or cause other adverse consequences. Thatis, bacteria are isolated if they are substantially free of bacteria ormaterials. Purity and homogeneity are typically determined usinganalytical techniques, for example, single cell culturing. The term“purified” can denote that a cell gives rise to essentially onepopulation.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms, especially of bacteria. That is,proliferation encompasses production of a greater number of bacteria,and can be measured by, among other things, simply counting the numbersof bacteria, measuring incorporation of ³H-thymidine into the bacteria,and the like.

As used herein, “sample” or “biological sample” refers to anything,which may contain the cells of interest (e.g., cancer or tumor cellsthereof) for which the screening method or treatment is desired. Thesample may be a biological sample, such as a biological fluid or abiological tissue. In one embodiment, a biological sample is a tissuesample including pulmonary arterial endothelial cells. Such a sample mayinclude diverse cells, proteins, and genetic material. Examples ofbiological tissues also include organs, tumors, lymph nodes, arteriesand individual cell(s). Examples of biological fluids include urine,blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinalfluid, tears, mucus, amniotic fluid or the like.

A “subject” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or improving an infectious disease or condition and/orone or more symptoms associated therewith. It will be appreciated that,although not precluded, treating an infectious disease or conditionand/or one or more symptoms associated therewith does not require thatthe disorder, condition, disease or symptoms associated therewith becompletely ameliorated or eliminated.

A “vector” is a composition of matter that comprises the Fcp1 gene andthat may be used to deliver the Fcp1 gene to the interior of a cell.Vector refers to any plasmid containing the Fcp1 gene that is capable ofmoving foreign sequences into the genomes of a target organism or cell.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of an embodiment for a variable or aspect herein includesthat embodiment as any single embodiment or in combination with anyother embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Leptospira

It has been discovered that flagellar-coiling protein 1 (Fcp1) plays anintegral role in Leptospira flagella. Functional loss of Fcp1 inLeptospira bacteria leads to motility deficiency and impaired bacterialvirulence. The invention includes, in one aspect, an isolated,flagellar-coiling protein 1 (Fcp1)-deficient Leptospira bacterium.

In one embodiment, the Leptospira bacterium comprises a silenced ordeleted Fcp1 gene. When silencing is employed, the expression of theFcp1 gene is prevented or decreased. An exemplary example of genesilencing is through a gene knockdown, such as RNAi. In anotherembodiment, the Leptospira bacterium comprises a mutant Fcp1 gene. Ininstances when the Fcp1 gene is mutated, the mutant Fcp1 gene mayexpress a mutant protein incapable of functioning as wildtype Fcp1.

Motility-deficient Leptospira bacteria are also included in theinvention. The isolated, Fcp1 deficient Leptospira bacteria possessedthinner periplasmic flagella than wildtype counterparts. The thinnerflagella lack tensile strength to generate sufficient thrust formotility of the bacterium. Motility is essential for penetration into ahost mucosa to establish infection. The invention also includes aLeptospira bacterium that has attenuated bacterial virulence. In anotherembodiment, the invention includes a Leptospira bacterium that isnon-pathogenic.

Another aspect of the invention includes a composition comprising aflagellar-coiling protein 1 (Fcp1)-deficient Leptospira bacterium. Inone embodiment, the composition includes the Leptospira bacteriumcomprising a silenced or deleted Fcp1 gene. In another embodiment, theLeptospira bacterium comprises a mutant Fcp1 gene. In some embodimentswhen the Leptospira bacterium includes a mutant Fcp1 gene, the mutantFcp1 gene expresses a mutant protein incapable of functioning aswildtype Fcp1 protein.

The invention also includes an embodiment where the compositioncomprises a Leptospira bacterium that is motility-deficient. In anotherembodiment, the Leptospira bacterium has attenuated bacterial virulence.In yet another embodiment, the Leptospira bacterium is non-pathogenic.

In another embodiment, the composition stimulates production of anantibody against the motility deficient Leptospira bacteria in thesubject. The antibody produced confers protection against infection by aheterologous pathogen, such as a different Leptospira bacteria, in thesubject or a new subject when transferred. The antibody produced can beof any class, such as IgG, IgM, or IgA or any subclass such as IgG1,IgG2a, and other subclasses known in the art. The antibodies may betransferred without purification, or isolated, purified, or otherwiseobtained from the original subject using methods known in the art. Theantibodies can then be administered to a second subject for protectionagainst Leptospira bacteria or a heterologous pathogen.

The invention also includes vaccines and compositions that can beformulated for use as vaccines. In one embodiment, a vaccine comprisesan effective amount of a motility deficient Leptospira bacteria. Inanother embodiment, a composition for stimulating an immune response ina subject in need thereof comprises an effective amount of a motilitydeficient Leptospira bacteria. The Leptospira bacteria of thecomposition is deficit in at lease one wild-type protein, such as, butnot limited to, flbB, flbD, flgA, flgB, flgC, flgD, flgG, flgH, flgI,flgM, flhA, flhB, flhF, flhX, fliA, fliE, fliF, fliG, fliG1, fliG3,fliH, fliI, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, motA, motA1,motB, motB, flgE, flgJ, flgK, flgL, flhO, fliD, fliK, fcp1, fcp2, flaA1,flaA2, flaB1, flaB2, flaB3, flaB4, fliS, and any combination thereof.The composition also includes a Leptospira bacterium that is a livebacterium. In another embodiment, the Leptospira bacterium isheat-inactivated. In yet another embodiment, the Leptospira bacteriumhas attenuated bacterial virulence.

The invention also includes a composition that further includes apharmaceutically acceptable carrier. In another embodiment, thecomposition further includes an adjuvant, such as an oil-in-wateremulsion, a saponin, a cholesterol, a phospholipid, a CpG, apolysaccharide, variants thereof, and a combination thereof.

Methods

The present invention also includes, in one aspect, a method ofproducing a motility-deficient Leptospira bacterium. As describedherein, the method comprises inhibiting expression of a wild-typeflagellar-coiling protein 1 (Fcp1) gene.

In one embodiment, inhibiting expression of the wild-type Fcp1 genecomprises deleting or silencing the wild-type Fcp1 gene in theLeptospira bacterium.

In another embodiment, inhibiting expression of the wild-type Fcp1 genecomprises mutating the wild-type Fcp1 gene in the Leptospira bacterium.In this and other embodiments, the mutant Fcp1 gene expresses a mutantFcp1 protein incapable of functioning as wildtype Fcp1 protein.

In another aspect, the invention includes a method of stimulating animmune response in a subject in need thereof comprising administering acomposition comprising an effective amount of a motility-deficientLeptospira bacteria to the subject. In yet another aspect, the inventionincludes a method for reducing or treating an infectious disease causedby one or more Leptospira bacteria in a subject in need thereofcomprising administering a composition comprising an effective amount ofa motility-deficient Leptospira bacteria to the subject. In oneembodiment, the Leptospira bacteria is deficit in a wild-type proteinthat functions in motility, such as but not limited to, flbB, flbD,flgA, flgB, flgC, flgD, flgG, flgH, flgI, flgM, flhA, flhB, flhF, flhX,fliA, fliE, fliF, fliG, fliG1, fliG3, fliH, fliI, fliJ, fliL, fliM,fliN, fliO, flip, fliQ, fliR, motA, motA1, motB, motB, flgE, flgJ, flgK,flgL, flhO, fliD, fliK, fcp1, fcp2, flaA1, flaA2, flaB1, flaB2, flaB3,flaB4, fliS, and any combination thereof.

In one embodiment, administering the composition comprises producingantibodies against the motility deficient Leptospira bacteria. Theproduction of antibodies may be short lived or long lasting within thesubject. Short lived antibody responses may be maintained over time byadministration of motility deficient Leptospira bacteria or boosts tothe immune response, such as through repetitive administrations of themotility deficient Leptospira bacteria. The antibodies generated mayconfer protection against infection by a heterologous pathogen, such asa different Leptospira bacteria, in the subject or or a new subject whentransferred. The antibodies generated can be of any class, such as IgG,IgM, or IgA or any, subclass such as IgG1, IgG2a, and other subclassesknown in the art. In another embodiment, the method further comprisesisolating the antibodies from the subject and transferring theantibodies to a new subject. The antibodies may be transferred withoutpurification, or isolated, purified, or otherwise obtained from theoriginal subject by methods known in the art. The antibodies may furtherbe administered to the second subject for protection against Leptospirabacteria or a heterologous pathogen. The administration of theantibodies may be through any methods known in the art of administeringantibodies.

Antigens that stimulate an immune response, yet do not producepathogenic disease in a subject, are exemplary vaccine candidates.Included in the methods of the invention are Leptospira bacteria thatcan stimulate an immune response, such as motility-deficient Leptospirabacteria. In one embodiment, the administered Leptospira bacteria arelive bacteria. In another embodiment, the Leptospira bacteria areheat-inactivated. In yet another embodiment, the Leptospira bacteriahave attenuated bacterial virulence. In yet another embodiment, theLeptospira bacteria are non-pathogenic.

The methods also include administering an adjuvant, separately or intandem with the compositions, such as an oil-in-water emulsion, asaponin, a cholesterol, a phospholipid, a CpG, a polysaccharide,variants thereof, and a combination thereof, with the composition of theinvention.

Pharmaceutical formulations that are useful in the methods of theinvention may be suitably developed for inhalational, oral, parenteral,pulmonary, intranasal, intravenous or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, and immunologically-based formulations. Theroute(s) of administration will be readily apparent to the skilledartisan and will depend upon any number of factors including the typeand severity of the disease being treated, the type and age of theveterinary or human patient being treated, and the like.

The pharmaceutical formulations described herein may be prepared by anymethod known or hereafter developed in the art of pharmacology. Ingeneral, such preparatory methods include the step of bringing the cellsinto association with a carrier or one or more other accessoryingredients, and then, if necessary or desirable, shaping or packagingthe product into a desired single- or multi-dose unit.

In one embodiment, the cells of the invention are formulated using oneor more pharmaceutically acceptable excipients or carriers. In oneembodiment, the pharmaceutical formulations of the cells of theinvention include a therapeutically effective amount of the cells of theinvention and a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers, which are useful, include, but are not limited to,glycerol, water, saline, ethanol and other pharmaceutically acceptablesalt solutions such as phosphates and salts of organic acids. Examplesof these and other pharmaceutically acceptable carriers are described inRemington's Pharmaceutical Sciences (1991, Mack Publication Co., NewJersey).

Administration/Dosing

In the clinical settings, delivery systems for the compositionsdescribed herein can be introduced into a subject by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical formulation of the composition can be administered byinhalation or systemically, e.g. by intravenous injection.

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the manifestation of symptoms associated withthe disease or condition. Further, several divided dosages, as well asstaggered dosages may be administered daily or sequentially, or the dosemay be continuously infused, or may be a bolus injection. Further, thedosages of the therapeutic formulations may be proportionally increasedor decreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the composition of the present invention to a subject,preferably a mammal, more preferably a human, may be carried out usingknown procedures, at dosages and for periods of time effective to treata disease or condition in the subject. An effective amount of thecomposition necessary to achieve a therapeutic effect may vary accordingto factors such as the extent of implantation; the time ofadministration; the duration of administration; other drugs, compoundsor materials used in combination with the composition; the state of thedisease or disorder; age, sex, weight, condition, general health andprior medical history of the subject being treated; and like factorswell-known in the medical arts. Dosage regimens may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation. Oneof ordinary skill in the art would be able to study the relevant factorsand make the determination regarding the effective amount of thecomposition without undue experimentation.

Actual dosage levels of the cells in the pharmaceutical formulations ofthis invention may be varied so as to obtain an amount of thecomposition that are effective to achieve the desired therapeuticresponse for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

Routes of Administration

Routes of administration of the compositions of the invention includeinhalational, oral, nasal, rectal, parenteral, sublingual, transdermal,transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral,vaginal (e.g., trans- and perivaginally), (intra)nasal, and(trans)rectal), intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topicaladministration.

Suitable formulation of the composition sand dosages include, forexample, dispersions, suspensions, solutions, beads, pellets, magmas,creams, pastes, plasters, lotions, discs, suppositories, liquid spraysfor nasal or oral administration, aerosolized formulations forinhalation, compositions and formulations for intravesicaladministration and the like.

It should be understood that the formulations and compositions thatwould be useful in the present invention are not limited to theparticular formulations set forth in the examples. The followingexamples are put forth so as to provide those of ordinary skill in theart with a complete disclosure and description of how to make and usethe cells, differentiation methods, engineered tissues, and therapeuticmethods of the invention, and are not intended to limit the scope ofwhat the inventors regard as their invention.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook,2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of AnimalCells” (Freshney, 2010); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1997); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Short Protocols in MolecularBiology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles,Applications and Troubleshooting”, (Babar, 2011); “Current Protocols inImmunology” (Coligan, 2002). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out embodiments of the presentinvention, and are not to be construed as limiting in any way.

The Materials and Methods used in the performance of the experimentsdisclosed herein are now described.

Bacterial Strains and Cloning Procedure.

The strains of L. interrogans used in this study, including the mutants,were grown to a mid log-phase in liquidEllinghausen-McCullough-Johnson-Harris (EMJH) medium (Ellinghausen etal., 1965, Am J Vet Res 26, 45-51) at 29° C.

In order to obtain clones for further studies in genetics ofleptospirosis, L. interrogans serovar Copenhageni strain LV 2756, avirulent clinical isolate recently cultivated from the blood of apatient with severe pulmonary hemorrhagic syndrome (SPHS) (Gouveia etal., 2008, Emerg Infect Dis 14, 505-508), and L. interrogans serovarCopenhageni strain Fiocruz L1-130 (Nascimento et al., 2004, Braz J MedBiol Res 37, 459-477), both enrolled in the hospital-based surveillancestudy in Salvador, Brazil (Ko et alk, 1999, Lancet 354, 820-825), wereplated in 1% agar plates of Ellinghausen-McCullough-Johnson-Harris(EMJH) medium (Ellinghausen et al., 1965, Am J Vet Res 26, 45-51;Johnson et al., 1967, J Bacteriol 94, 27-31), in a concentration of 100and 10 leptospires, after determination of the number of cells per mLusing a Petroff-Hausser counting chamber (Fisher Scientific). Plateswere incubated at 29° C., and visualized weekly during a period of 4weeks. The colonies obtained were cultivated in liquid EMJH at 29° C.,and observed under dark-field microscopy.

E. coli strains were grown in Luria-Bertani (LB) medium. When necessary,spectinomycin and/or kanamycin were added to culture media at aconcentration of 50 μg/mL.

Genomic DNA was extracted from a pellet of 5 mL cultures using theMaxwell®16 (Promega Corporation, Madison, Wis.).

Construction of Mutant and Complemented Strain.

Mutagenesis experiments were performed using the conjugation method(Picardeau, 2008, Appl Environ Microbiol 74, 319-322), and for coloniesselection, 1% agar plates of EMJH. Strains of E. coli used for themutagenesis experiments were grown in Luria-Bertani (LB) medium. Whennecessary, spectinomycin and/or kanamycin were added at a concentrationof 50 μg/mL in both culture media.

For allelic exchange of the fcp1 gene, an upstream and downstream regionof the gene were amplified from the genomic DNA of L. interrogansserovar Copenhageni strain Fiocruz L1-130 using primers Fcp1_FlkAF andFcp1_FlkAR for the upstream region, and Fcp1_FlkBF and Fcp1_FlkBR forthe downstream region. See Table 1. The PCR products of upstream anddownstream region were digested with BamHI and XbaI, and HindIII andSpeI, respectively. The Spcr cassette was amplified using primersSpc_Xba5 and Spc_Hind3, and the PCR product was digested with XbaI andHindIII. The three digested PCR products were transformed into thenon-replicative plasmid pSW29T (Picardeau, 2008, Appl Environ Microbiol74, 319-322), previously digested with BamHI and SpeI. The finalplasmid, containing the flanking regions of the fcp1 gene, which wasreplaced by the Spcr cassette, was transfected into the donor strain E.coli (32163 cells, and introduced into strain Fiocruz L1-130 byconjugation, as previously described (Picardeau, 2008, Appl EnvironMicrobiol 74, 319-322). After 4 to 6 weeks of plate incubation at 30°C., spectinomycin-resistant transformants were inoculated into liquidEMJH supplemented with 50 μg/ml of spectinomycin, and examined forallelic exchange in the target gene by PCR, using primers Fcp1_AscF andFcp1_AscR, and by Western blotting. See Table 1.

TABLE 1 Sequence of primers used in this study Primer Sequence (5′-3′)SEQ ID NO Fcp1_FlkAF CGGGATCCCGGATTTCTTGGGTCATTTCTT SEQ ID NO: 3Fcp1_FlkAR GCTCTAGAGCTTCTCTTTCAATGGTATTAG SEQ ID NO: 4 Fcp1_FlkBFCCCAAGCTTGGGCGTTCACCTTTTGAGAGCGA SEQ ID NO: 5 Fcp1_FlkBRGGACTAGTCCGCTTCAATCGACCGTTTCCA SEQ ID NO: 6 Spc_Xba5GCTCTAGAAACGCGTCCCGAGC SEQ ID NO: 7 Spc_Hind3CCCAAGCTTAACGCGTAAAGTAAGCACC SEQ ID NO: 8 Fcp1_AscFGGCGCGCCTGGATCATTGAATAGTCTAT SEQ ID NO: 9 Fcp1_AscRGGCGCGCCAAGGATCTTGGTTCGTAAAA SEQ ID NO: 10 LipL32-45FAAGCATTACCGCTTGTGGTG SEQ ID NO: 11 LiL32-286R GAACTCCCATTTCAGCGATTSEQ ID NO: 12 LipL32-189p [6-FAM]AAAGCCAGGACAAGCGCCG[BHQ1a-Q]SEQ ID NO: 13 GAPDH_R GGTTCACACCCATCACAAACAT SEQ ID NO: 14 GAPDH_FGGTGGAGCCAAGAGGGTCAT SEQ ID NO: 15 GAPDH_P[6-FAM]ATCTCCGCACCTTCTGCTGATGCC SEQ ID NO: 16 [BHQ1a-Q]

For complementation, the fcp1 gene together with its predicted promoter,was amplified with primers Fcp1_AscF and Fcp1_AscR from L. interrogansstrain Fiocruz L1-130. The amplified product, after digestion with AscI,was cloned into the suicide pSW29T_TKS2 plasmid (Picardeau, 2008, ApplEnviron Microbiol 74, 319-322), which carries a kanamycin-resistantHimar1 transposon. Random insertion mutagenesis by conjugation wascarried out in L. interrogans strain Fiocruz L1-130 fcp1⁻ and strain LV2756 motility deficient, as previously described (Murray et al., 2009,Infect Immun 77, 810-816). Semi-random PCR was used for identificationof the transposon insertion site (Murray et al., 2009, Infect Immun 77,810-816).

For whole-cells lysates, PFs purification and Cryo-ET experiments,Leptospira cultures were grown to a late log-phase and prepared aspreviously described (Lourdault et al., 2011, Infect Immun 79,3711-3717; Trueba et al., 1992, J Bacteriol 174, 4761-476). Pictures andvideos were done under a Zeiss Axiolmager. M2 dark field microscopy withAxioCam MRm camera, and analyzed using the AxioVision 4.8.2 software(Carl Zeiss Microscopy LLC).

Flagella Preparation.

Purification of the periplasmic flagella (PFs) was performed bymodification of a previous reported protocol (Trueba et al., 1992, JBacteriol 174, 4761-476). Briefly, 300 mL of a broth culture oflate-logarithmic-phase cells (approximately 5×10⁸ cells/ml) wereharvested and centrifuged at 8,000×g for 20 min at 4° C. The cell pelletwas resuspend and washed in 28 mL of with PBS. The cell pellet was thenresuspend in 30 mL of sucrose solution (0.5 M sucrose, 0.15 M Tris, pH8.0), and centrifuged at 8,000-×g for 15 minutes. The pellet wasresuspend in 8 mL of sucrose solution, and stirred on ice for 10minutes. To remove the spirochete outer membrane sheath, 0.8 mL of a 10%Triton X-100 solution (1% final concentration) was added and the mixturewas stirred for 30 minutes at room temperature, and 80 μL solution ofLysozyme (10 mg/mL) was added slowly and stirred on ice for 5 minutes.Before a 2 h stirring at room temperature, 0.8 mL of EDTA solution (20mM, pH 8.0) was added slowly. After, 160 μL of MgSO₄ solution (0.1M),and 160 μL of EDTA solution (0.1M, pH 8.0) were added, both withintervals of 5 minutes with stirring at room temperature. The suspensionwas centrifuged at 17,000×g for 15 minutes, and the supernatant fluidwas mixed well with 2 mL of PEG 8000 solution (20%) in 1M NaCl), andkept on ice for 30 minutes. After centrifugation at 27,000×g for 30minutes, the pellet was resuspend in 3 mL of H₂O, and a newcentrifugation was performed, at 80,000×g for 45 minutes. The finalpellet, consisting of purified PFs, was suspended in 1 mL of H₂O andstored at 4 C.

Gel Electrophoresis, Immunobloting and Protein Analysis.

SDS/PAGE and Western blotting were carried out as previously described((Lourdault et al., 2011, Infect Immun 79, 3711-3717). Antibodiesα-FlaA1 were used as positive control in the western blot of themutants. Cell lysates, and polyclonal antibodies were prepared asdescribed (Croda et al., 2008, Infect Immun 76, 5826-5833). For thewestern-blot with vaccinated animals, a pool of sera from hamsters 21days after vaccination with the L. interrogans fcp1⁻ and a pool of serafrom hamsters vaccinated with heat-killed bacteria was used as primaryantibodies against whole-cell extracts of L. interrogans Fiocruz L1-130,Manilae, Canicola and Pomona.

Mass spectrometry analysis (MS+MS/MS) of the whole cell lysates and PFspreparation of the L. interrogans strain LV 2756 motile and strain LV2756 motility deficient were carried out from fragments of thepolyacrylamide gel stained with coomassie blue, according to protocolsof the Proteomics Platform of the Institute Pasteur, Paris, France. Twoindependent experiments for each sample and strain were performed.

Electron Microscopy.

Culture in late log-phase (5 mL) was centrifuged at 3.000 rpm for 15 minat 4° C. The supernatant was removed and 5 mL of fixative containing 2%glutaraldehyde and sodium cacodylate buffer (pH 7.4) 0.1M was added tothe pelleted cells. The cells were fixed for 1 h at 4° C. and thenplaced on coverslips treated with poly-L-lysine. After this step, thecells were post-fixed with 1% osmium tetroxide and treated with a gradedseries of ethanol solutions. The samples were critical point drying,sputter coating with gold and examined using a JEOL 6390LV scanningelectron microscopy (SEM).

Preparation of purified PFs (10 μL) was allowed to adsorb for 60 s ontoa copper grid coated with Formvar 400 mesh. The grid was washed threetimes with 0.1M sodium cacodylate and then negatively stained with 2%(w/v) phosphotungstic acid (PTA) pH 7.2. Grids were observed using aJEOL JEM1230 transmission electron microscope (TEM) operating at 80 keV.

For the diameter measurement of the flagella, twenty random pictureswere taken from each group on the same magnification (200.000×), using aGatan camera and software DigitalMicrograph® for acquisition. Fourdifferent measurements of the PFs thickness were taken from each strain,using the ImageJm1.45s software. The averages of those measurements wereused for comparison between groups.

Immuno-EM Assays.

Purified PFs (15 μL) were allowed to adsorb for 10 min inglow-discharged copper grids coated with Formvar 300 mesh. Immediatelythe grids were blocked for 2 min in 0.1% BSA, and incubated for 20 minwith 1:10 dilution (0.1% BSA) of primary antibody. Polyclonal antibodiesanti-FlaA1, FlaA2, anti-FlaB1, and Fcp1 were used as primary antibodies.The grids were washed 3 times with ultrapure water, and blocked again in0.1% BSA for 2 min. Secondary antibody 5 nm gold-conjugated Protein A(PAG) was used in a dilution of 1:50 (0.1% BSA), incubated for 20 min.The grids were washed three times with ultrapure water and thennegatively stained with 2% PTA pH 7. Grids were observed using a PhilipsTECNAI 12 BioTwin II operating at 80 keV. Images were acquired on SoftImaging System Morada camera using iTEM image acquisition software.

Cryo-Electron Tomography and 3-D Reconstruction.

Viable bacterial cultures were centrifuged to increase the concentrationto ˜2×10⁹ cells/nil. Five-microliter samples were deposited onto freshlyglow-discharged holey carbon grids for 1 min. The grids were blottedwith filter paper and rapidly frozen in liquid ethane using agravity-driven plunger apparatus as previously described (Raddi et al.,2012 J Bacteriol 194, 1299-1306). The resulting frozen-hydratedspecimens were imaged at −170° C. using a Polara G2 electron microscope(FEI Company, Hillsboro, Oreg.) equipped with a field emission gun and a4K×4K charge-coupled-device (CCD) (16-megapixel) camera (TVIPS; GMBH,Germany). The microscope was operated at 300 kV with a magnification of×31,000, resulting in an effective pixel size of 5.6 Å after 2×2binning. Using the FEI batch tomography program, low-dose single-axistilt series were collected from each bacterium at −6 μm defocus with acumulative dose of ˜100 e⁻/Å² distributed over 87 images, covering anangular range from −64° to +64°, with an angular increment of 1.5°.Tilted images were aligned and then reconstructed using IMOD softwarepackage. In total, 10, 15 and 17 reconstructions were generated from WT,fcp1 mutant and complemented strains, respectively.

A total of 1392 segments (192×192×96 voxels) of flagellar filaments weremanually identified and extracted from 42 reconstructions. The initialorientation was determined using two adjacent points along the filament.Further rotational alignment was performed to maximize thecross-correlation coefficient. Averaging was carried out with a mergingprocedure in reciprocal space (Raddi et al., 2012 J Bacteriol 194,1299-1306).

Tomographic reconstructions were visualized using IMOD. Reconstructionof cells were segmented using 3D modeling software Amira (VisageImaging). Three-dimensional segmentations of the cytoplasmic, outermembranes and flagellar filaments were manually constructed.

Translocation Assays with MDCK Cells.

A translocation assay was performed according to a protocol modifiedfrom that described by Figueira et al. (Figueira et al., 2011, BMCMicrobiol 11, 129). MDCK cells at a concentration of 2×10⁵ cells in 500μl of DMEM were seeded onto 12-mm-diameter transwell filter units with3-μm pores (COSTAR). Monolayers were incubated at 37° C. in 5% CO2 for 3to 4 days with daily changes in media until the transepithelialresistance (TER) reached a range of 200 and 300 Ω/cm2, as measured withan epithelial voltohmmeter (EVOM, World Precision Instruments, Sarasota,Fla.). The TER for polycarbonate filters without cells was approximately100 Ω/cm2. The upper chamber of the transwell apparatus was inoculatedwith a multiplicity of infection (MOI) of 100 leptospires by adding 500μL of bacteria, which were resuspend in 1:2 v/v ratio of DMEM and EMJHmedia. Duplicate transwell chamber assays were performed for eachleptospiral strain tested. Aliquots were removed from lower chamber (100μl) at 2, 4, 6 and 24 hours and the number of leptospires were countedin triplicate by using the Petroff-Hausser counting chamber (FisherScientific). The ability of leptospires to translocate MDCK polarizedmonolayers was determined by calculating the proportion of leptospiresin the lower chamber in comparison to the initial inoculums forduplicate assays at each time point.

Virulence Studies.

All hamster experiments used 3-6 week-old Golden Syrian male hamstersfrom Harlan Laboratories, and were conducted following the NationalInstitutes of Health guidelines for housing and care of laboratoryanimals. All the procedures were performed under animal protocolsapproved by the Yale University Institutional Animal Care and UseCommittee and Gonçalo Moniz Research Center, Fiocruz. The bacterialchallenges in animals were performed using doses from 10-10⁸ leptospiresby IP route and dose of 10⁸ leptospires by CJ route.

For the experiments of virulence, one group of 8-10 animals for each ofthe six strains was inoculated intraperitoneally (IP) with a high-doseinoculum (10⁸ leptospires) in 1 ml of EMJH medium. For the LD50experiments, two groups of 4 animals were inoculated IP with doses of100 and 10 leptospires, for the strains LV2756 motile, LV2756motility-deficient fcp1⁺, Fiocruz L1-130 WT and Fiocruz L1-130fcp1^(−/+). For the strains LV2756 motility-deficient and Fiocruz L1-130fcp1⁻, animals were infected with doses of 10⁸ and 10⁷ leptospires.

Animals were monitored twice daily for clinical signs of leptospirosisand death, up to 21 days post-infection. Moribund animals wereimmediately sacrificed by inhalation of CO₂.

Dissemination Studies.

For the experiments of dissemination, one group of six animals forstrains LV2756 motile and LV2756 motility deficient was inoculatedintraperitoneally with 10⁸ leptospires in 1 ml of EMJH medium. After 1hour and 4 days post-infection, sub-groups of two animals wereeuthanized. With the same strains, a conjunctival infection wasperformed by centrifugation of 30 ml culture of leptospires for 10minutes at 1000 rcf and using an inoculum of 10⁸ leptospires in 10 μl ofEMJH medium instilled in the left eye conjunctiva using a micropipette.Groups of four animals were infected and two were euthanized after 7days of infection for each strain tested. In those experiments, a groupof two animals was left as a positive control.

To understand the dissemination of the fcp1⁻ mutant using a subcutaneous(SC) route, we infected groups of 10 animals with 10⁷ bacteria of L.interrogans Fiocruz L1-130 wild-type, fcp1⁻ mutant and heat-killedwild-type by subcutaneous route. For each group of infection two animalswere euthanized after 1, 4, 7, 14 and 21 days post-infection.

Animals were monitored twice daily for clinical signs of leptospirosisand death, up to 21 days post-infection. Moribund animals wereimmediately sacrificed by inhalation of CO₂.

The necropsy for the dissemination study was made as follows. Animalswere sacrificed by inhalation of CO₂ and placed on their backs slightlyinclined in the dissecting tray. After sterilization of the abdomen withalcohol 70% and using sets of sterile instruments, the internal organswere exposed, including the heart and lungs. All blood was collecteddirectly from the heart in a Vacutainer® K2 EDTA Tubes (BD Diagnostics,Franklin Lakes, N.J.) and glass serum tubes, using a 5 ml syringe with a21 G needle. A 21 G butterfly needle affixed to a 60 ml syringecontaining sterile saline 0.85% was then inserted into the leftventricle. The right atrium was snipped to allow the residual blood andnormal saline to leave the body during the perfusion. Each hamster wasperfused with 100 ml of saline solution. After perfusion, rightpulmonary lobe, right dorsocaudal hepatic lobe, spleen, right kidney andright eye were carefully removed. All the tissues were collected intocryotubes and immediately placed into liquid nitrogen before beingstored at −80° C. until extraction. Blood, kidney, liver, lung, spleenand eye were analyzed.

Using scissors and scalpels, 25 mg of lung, liver, kidney cortex, andeye, 10 mg of the spleen, and 200 μl of blood were asepticallycollected. DNA was extracted using the Maxwell® 16 Tissue DNApurification kit (Promega Corporation, Madison, Wis.), afterhomogenization with Bullet Blender (Next Advance, Averill Park, N.Y.).

Quantitative Real Time PCR.

Bacterial quantification was determined using an ABI 7500 (AppliedBiosystems, Foster City, Calif.) and Platinum Quantitative PCRSuperMix-UDG (Invitrogen Corporation, Carlsbad, Calif.). The lipL32 genewas amplified using a of primers and probe (Table 1), according toprotocol previously described (Stoddard et al., 2009, Diagn MicrobiolInfect Dis 64, 247-255). Hamster housekeeping geneglyceraldehyde-3-phophate dehydrogenase (GAPDH) was used as a controlfor PCR inhibitors and to monitor nucleic acid extraction efficiency.The forward primer of GAPDH_F and GAPDH_R were selected to amplify afragment that was detected by the probe, GAPDH_P. A sample with athreshold cycle (Ct) value between 16 and 21 was considered as positiveand further analyzed by real-time PCR targeting lipL32. In case of anegative sample, a new DNA extraction was performed. For each organ, theDNA was extracted from one sample and the Real Time PCR was performed induplicate. Considering the amount of tissue that was used for DNAextraction, an equation was applied to express the results as the numberof leptospires per gram of tissue or per ml of blood/water.

Immunization Studies.

Groups of 7-10 hamsters were immunized with 10⁷ bacteria with L.interrogans Fiocruz L1-130 wild-type, motility-deficient fcp1⁻ mutantand heat-killed bacteria by SC route and challenged 21 days later with alethal dose of a range of serovars whose virulence has beenwell-characterized in our laboratory (Table 2). Animals were infected byconjunctival inoculation, which mimics the natural route of infection,with a dose of 10⁸ bacteria. Animals were monitored twice daily forclinical signs of leptospirosis and death, up to 21 days post-infection.Moribund animals were immediately sacrificed by inhalation of CO₂.

TABLE 2 Strains used in vaccine experiments Species Serovar Strain LD₅₀*L. interrogans Copenhageni Fiocruz L1-130 <10 L. interrogans CanicolaKito <10 L. interrogans Pomona PO-06-047 <10 L. interrogans Manilae L495<10 L. kirschneri Grippotyphosa RM52 <10 *LD₅₀ for intraperitonealinoculation of hamsters

Passive transfer experiments. Golden Syrian Hamsters were immunized withone dose of the L. interrogans Fioruz L1-130 fcp1⁻ live-attenuatedvaccine, and bled the animals after 21 days to obtain hyper-immune sera.Then 3 mL of the hamster sera was transferred intraperitoneally (IP) toa group of 3 naïve hamsters 16 hours prior to lethal challenge of 10⁷leptospires by IP route in with the heterologous strain L. interrogansManilae. A control group of 3 hamsters were passive-transferred with 3mL of sera from non-vaccinated animals. Animals were monitored twicedaily for clinical signs of leptospirosis and death, up to 14 dayspost-infection. Moribund animals were immediately sacrificed byinhalation of CO₂.

Statistical Analysis.

Data were graphed and analyzed using GraphPad Prism 5.0c (GraphPadSoftware, La Jolla, Calif.). Fisher's exact test and analysis ofvariance (ANOVA) were performed to assess statistical significance ofdifferences between pairs of groups and multiple groups, respectively. Pvalues <0.05 were considered to be significant.

The Results of the experiments disclosed herein are now described.

Identification of a L. interrogans Motility-Deficient Mutant.

The cloning process of L. interrogans serovar Copenhageni strain 2756 insolid EMJH yielded two distinct sizes of colony growth (FIG. 1A). Largecolonies, identified after 3 weeks of incubation of the plates at 30°C., had the same morphological shape and motility characteristics as theparental strain when observed by dark field microscopy (FIG. 1B),whereas the small colonies, identified after 4 weeks of incubation had alinear appearance, lacking the characteristic hook-shaped end ofleptospires, had no translational motility, and grew as long chains withincomplete division planes (FIG. 1B). Analysis under scanning electronmicroscopy showed that the motility-deficient cells retained theirspiral body morphology (FIG. 1C).

Negative stain electron microscopy of purified PFs from both clonesshowed that the motility-deficient clone had straight flagella,different from the characteristic extensive coiled flagella of themotile clone (FIG. 1D).

A Novel Protein Involved in Leptospiral Motility.

SDS-PAGE of whole cell lysates and PFs preparations from the motileclone of L. interrogans strain LV 2756 showed a band of 36 kDa thatlacks in the strain LV 2756 motility-deficient clone (FIG. 2B). Massspectrometry (MS) analyses of this 36 kDa protein band from the motileclone showed the presence of 7 and 4 different peptides in whole celllysates and purified PFs, respectively. One of these peptidescorresponded to a hypothetical protein coded by the gene LIC13166 fromL. interrogans serovar Copenhageni strain Fiocruz L1-130. Similaranalyses using SDS-PAGE gel fragments around the 36 kDa region withproteins obtained from whole cell lysates and purified PFs from themotility-deficient clone revealed no peptide corresponding to LIC13166.

This gene is present in all Leptospira species sequenced so far,including the saprophyte one, L. biflexa serovar Patoc, which has anorthologous gene with 76% nucleotide identity. The protein encoded byLIC13166 has been described as the 13^(th) most abundant among all cellproteins in L. interrogans (Malmstrom et al., 2009, Nature 460,762-765). However, no orthologous genes outside the Leptospiraceaefamily are known. It has a predicted signal peptide (first 25 aminoacids), and it was previously described as an outer membrane, OmpL36(Pinne and Haake, 2009, PLoS One 4, e6071) and a putative coagulaseinvolved in the pathogenesis. More recently, it was showed that it hasno ligand-binding activity against the main host-tissue components, butit is recognized by acute and convalescent leptospirosis patients' seraas well as by sera from hamsters infected with leptospires.Two-dimensional gel electrophoresis analyses showed that it wasoverexpressed under in vivo-like conditions (iron limitation withpresence of fetal bovine serum), and also showed higher levels ofexpression in pathogenic serovars. Given the correlation of itsexpression with the normal structure of leptospiral PFs, the proteinencoded by LIC13166 was renamed as Flagellar coiling protein (Fcp1),predicted to have 306 amino acids.

Purified PFs from the motile clone showed the expression of Fcp1 asrevealed by western blot analyses using a monospecific anti-Fcp1polyclonal antibody (FIG. 2B). In contrast, no Fcp1 expression wasdetected in the motility-deficient clone, neither in whole-cell lysatesnor purified PFs (FIG. 2B), indicating that the spontaneous mutationabolished expression of Fcp1 protein in this clone. DNA sequencing ofthe fcp1 gene in both mutants showed an insertion of a deoxythymidine inthe position 3876851 (corresponding to amino acid 286) of the L.interrogans serovar Copenhageni strain Fiocruz L1-130 genome,introducing a reading frame shift in the motility-deficient clone genethat lead to a premature stop codon at amino acid position 294 (FIG. 3).

Allelic Exchange Mutagenesis and Complementation.

To confirm that the phenotype observed in the motility-deficient clonewas caused by the mutation in the fcp1 gene, a gene replacementconstruct was generated by homologous recombination (FIG. 3), whichcould be confirmed by PCR. Allelic exchange resulted in the null mutantFiocruz L1-130 fcp1⁻, which has a motility-deficient phenotype whencompared to parental strain, Fiocruz L1-130 WT.

Complemented strains were obtained by random mutagenesis using Himar1transposon carrying the fcp1 gene from L. interrogans strain FiocruzL1-130. By semi-random PCR, the transposon insertion sites in 4transformants were identified for the motility-deficient strain LV 2756,eventually selecting the clone LV 2756 motility-deficient fcp1⁺. Thismutant had the transposon inserted in a non-coding region between thegenes LIC12898 and LIC12899, which encodes for a hypothetical and acytoplasmic membrane protein, respectively.

For the strain Fiocruz L1-130 fcp1⁻, 3 transformants were obtained, andthe clone Fiocruz L1-130 fcp1^(−/+) was selected, with the transposoninserted in a non-identified region. However, sequencing results bysemi-random PCR allowed designing primers that confirmed the insertionof the transposon, identifying several stop codons for the 6 frames inthe region, and indicated that the transposon was inserted in anon-coding region.

SDS/PAGE and Western blot analyses showed that the fcp1 mutant lacks theexpression of Fcp1, and that the complementation was able to rescue itsnormal expression (FIG. 2). Analysis of the mutant Fiocruz L1-130 fcp1⁻by motility assay, dark field and electron microscopy confirmed that itsphenotype was identical to the LV 2756 motility-deficient clone.Furthermore, expressing Fcp1 in the mutants restored the hook-shaped endof the cells, and their normal translational motility as seen by theanalyses of both complemented strains (FIG. 1A-C). Purified PFs obtainedfrom Fiocruz L1-130 fcp1⁻ had the same straight phenotype as that of theLV 2756 motility-deficient clone, and the coiled morphology was restoredby the complementation of the fcp1 gene, resulting in identical featuresas observed in the LV 2756 motile and Fiocruz L1-130 WT clones (FIG.1D). Fcp1 was detected in purified PFs preparation in Fiocruz L1-130 WTand complemented mutant strain, confirming its localization in flagella.Altogether these findings indicate that the lack of Fcp1 was responsiblefor the disappearance of the flagellum coil structure associated with aloss of cell motility.

Fcp1 is Necessary for the Formation of the PFs Sheath in LeptospiraInterrogans.

The diameter of the purified PFs was analyzed from all six differentstrains obtained, to determine if Fcp1 was involved in the structure ofleptospiral flagella. Four different in-vitro measurements were madefrom each of the twenty images of the PF of each strain. The averagediameter of PFs purified from the LV 2756 motile and Fiocruz L1-130 WTclones were 21.5±1.99 nm, and 22.8±2.01 nm, respectively, whereas thatof the PFs purified from the LV 2756 motility-deficient and L1-130 fcp1⁻clones were 16.27±2.88 nm, and 17.63±0.92 nm, respectively. Thedifferences between the wild-type and the mutants' diameters werestatistically significant (p<0.0001). Expressing Fcp1 in fcp1⁻ mutantsallowed the restoration of the wild-type average diameter of the PFs,showing no significant difference with respect to the wild-type.

In order to examine the cellular morphology and the flagellar structureof the wild-type, fcp1⁻ mutant and complemented strain,three-dimensional reconstructions in situ of intact organisms weregenerated using cryo-electron tomography (cryo-ET). The resultsconfirmed that the flagellar filament of the fcp1⁻ mutant wassignificantly thinner than that those in the wild-type and complementedstrain (FIG. 4).

Immuno-EM assays using antibodies anti-Fcp1 indicated that this proteinis being expressed on the surface of the PFs, evenly distributed alongits whole length in the Fiocruz WT strain (FIG. 5). The fcp1-mutantdidn't show any expression of the Fcp1 protein. Antibodies anti-FlaB1,anti-FlaA1 and anti-FlaA2 were used as control, however there was nobinding in either of the two strains used (FIG. 5). This assay indicatesthat the lack of antibody binding Fcp1 protein is because no Fcp1proteins were surfaced exposed.

Attenuated, Motility Deficient Strains are Unable to Translocate AcrossTissue Barriers.

The lack of expression of Fcp1 leads to a reduced ability to translocatea monolayer of MDCK cells, as showed by the results obtained with thetranslocation assay (FIG. 7). The strain Fiocruz L1-130 fcp1⁻ was unableto translocate across the monolayer of MDCK cells after 24 h ofincubation, while after 2 h of incubation 0.6% of both Fiocruz L1-130 WTand Fiocruz L1-130 fcp1^(−/+) were recovered, reaching a recovery rateof 25.5% and 46.7%, respectively, after 24 h of incubation.

Motility is Essential for Leptospiral Virulence.

To determine if Fcp1 plays a role in the pathogenesis of the disease,groups of 8-10 hamsters were infected intraperitoneally (IP) with 10⁸leptospires (in three independent experiments), with all the 6 strainsdescribed in this study (Table 3). The strains LV 2756 motile andFiocruz L1-130 WT clones were able to kill all animals between the6^(th) and 8^(th) day post-infection (Table 3).

In three experiments, animals challenged with strain LV 2756motility-deficient clone survived after 21 days post-infection. Allanimals infected with strain Fiocruz L1-130 fcp1⁻ survived in two of theexperiments (Table 3), but 50% died in one of the experiments, betweendays 6 and 10 post-infection. The complementation of the fcp1 gene wasable to restore the phenotype of virulence, and animals challenged withstrains LV 2756 motility-deficient fcp1⁺ and Fiocruz L1-130 fcp1^(−/+)were able to kill 100% of the animals between days 6 and 10post-infection (Table 3).

TABLE 3 Virulence of strains of L. interrogans serovar Copenhageni afterhamster where inoculated intraperitoneally with 10⁸ leptospires^(§)Mortality Bacterial Strain (%) Time to death (days) Fiocruz LV2756motile 100 6, 6, 6, 6, 6, 6, 6, 8 Fiocruz LV2756 motility-deficient  0*— Fiocruz LV2756 motility-deficient 100 6, 6, 8, 8, 9, 9, 10, 10 fcp1⁺Fiocruz L1-130 WT 100 6, 6, 6, 8, 8, 8, 8, 8 Fiocruz L1-130 fcp1⁻ 0^(¶)* — Fiocruz L1-130 fcp1^(−/+) 100 6, 6, 6, 8, 8, 8, 8, 8^(§)Results are showed for one of three independent experiments. Groupof 08 animals were challenged using an intraperitoneal infection dose of10⁸ leptospires. All the experiments showed equivalent results.^(¶)Mortality rate of 50% in one experiment for this specific strain (p= 0.038) *Statistically significance difference when compared withwild-type results (p = 0.00007)

To assess the difference showed between strains Fiocruz L1-130 fcp1⁻ andthe LV 2756 motility-deficient strains, and also to determine if thecomplemented strains were still highly virulent as the wild-type ones,two independent experiments of LD₅₀ were performed, with two groups offour hamsters inoculated by the IP route. Animals challenged with thestrains LV 2756 motile, LV 2756 motility-deficient fcp1⁺, Fiocruz L1-130WT and Fiocruz L1-130 fcp1⁻ were infected with doses of 100 and 10leptospires, and animals challenged with the LV 2756 motility-deficientand Fiocruz L1-130 fcp1⁻ strains were infected with doses of 10⁸ and 10⁷leptospires. As previously described (Croda et al., 2008, Infect Immun76, 5826-5833), the LD₅₀ for the strain Fiocruz L1-130 WT was lower than10 leptospires, and the same result was found for the strain LV 2756motile and the two complemented strains, while the LV 2756motility-deficient and Fiocruz L1-130 fcp1⁻ strains had a LD₅₀ of >10⁸leptospires and 10⁸ leptospires respectively (Table 4), showing thatmotility is paramount for virulence, and Fcp1 plays a key role.

TABLE 4 LD⁵⁰ of L. interrogans serovar Copenhageni after hamsters wereinoculated intraperitoneally with 10⁸ leptospires^(§) Bacterial StrainLD⁵⁰ Fiocruz LV2756 motile 4.64 leptospires Fiocruz LV2756motility-deficient >10⁸ leptospires^(¶) Fiocruz LV2756motility-deficient fcp1⁺ 4.64 leptospires Fiocruz L1-130 WT <10leptospires* Fiocruz L1-130 fcp1⁻ 10⁸ leptospires Fiocruz L1-130fcp1^(−/+) 10 leptospires ^(§)Results are showed for one of threeindependent experiments. Three groups of 4 animals for each strain werechallenged using an intraperitoneal infection dose of 10⁸ leptospires.All the experiments showed equivalent results. ^(¶)All the animalsinfected with 10⁸ leptospires survived *All the animals infected with 10leptospires died

Motility-Deficient Strains, Although Attenuated, Induces a TransientBacteremia after Experimental Infection.

To determine the dissemination of leptospires in the hamster model,quantitative real time PCR was performed in tissues of animals infectedwith 10⁸ leptospires using the IP and conjunctival (CJ) inoculationroute. Groups of IP infected hamsters were euthanized 1 hour, and 4 daysafter infection.

All control animals infected with the strain LV 2756 motile clone diedafter 5-6 days of infection, while the animals infected with the strainLV 2756 motility-deficient survived after 30 days post-infection. Alltissues of strain LV 2756 motile analyzed were positive after 1 hour forthe presence of the agent, with a range of 2×10³-5×10⁴ leptospires/g oftissue, while the strain LV 2756 motility-deficient were undetectable(FIG. 6A). After 4 days of infection, the burden of the strain LV 2756motile ranged from 1.5×10⁴ to 6×10⁸ leptospires/g in the eye and liver,respectively. Although after 4 days post-infection, leptospires weredetectable in all the tissues when inoculated with strain LV 2756motility-deficient, the burden of leptospires was significantly lower incomparison with the motile clone, ranging from 1.7×10⁵ to 5×10² in thespleen and liver, respectively (FIG. 6B). Analysis after 30 days oftissues from animals that survived of the LV 2756 motility-deficientchallenge showed no detectable leptospires in any tissue, and there wasno detection of leptospires in the eye of infected animals at any timepoint (FIG. 6A-B).

When hamsters were infected with 10⁸ leptospires by CJ route, none ofthe analyzed tissues infected with the strain LV 2756 motility-deficientwere positive for the presence of leptospires after seven dayspost-infection (FIG. 6C), while for the LV 2756 motile all the tissueswere positive, with a burden ranging from 4×10³ leptospires/g in the eyeto 2×10⁶ leptospires/g in the kidney (FIG. 6C), with the control animalsdying after 8-9 days post-infection. All the control animals infectedwith the LV 2756 motility-deficient survived, and there was no detectionof leptospiral DNA after 30 days in any of the tissues.

Although the motility-deficient mutant has a strong phenotype ofattenuation, it was still able to cause a transient systemic infectionafter IP (FIG. 6B) and conjunctival (FIG. 8) infection, which wascleared 7 days after inoculation (FIG. 8). There was no evidence of DNAdissemination in any tissues after SC vaccination with heat-killedbacteria (FIG. 8).

Immunization with Attenuated, Motility-Deficient fcp1-Strains ConfersProtection Against Leptospirosis Due to Homologous and HeterologousSerovars.

It was also determined if motility-deficient fcp1-mutant may serve as acandidate for a live attenuated vaccine since: 1) deletion of thefcp1-gene renders mutants unable to produce disease or renalcolonization, 2) the transient bacteremia produced by needle inoculationmay be sufficient to induce robust and long-lasting immune responses,and 3) attenuated leptospires may elicit cross-protective immuneresponses against protein moieties conserved across pathogenicLeptospira. This was evaluated by administering hamsters with a singlesubcutaneous dose of heat-killed, wild-type bacteria and livefcp1-mutant of L. interrogans serovar Copenhageni, and then infectinghamsters three weeks after immunization with a lethal dose of fourserovars of L. interrogans species, serovars Copenhageni, Manilae,Canicola and Pomona, and the serovar Grippotyphosa, which belongs to L.kirschneri, a different pathogenic species of the Leptospira genus.

As expected, immunization with heat-killed bacteria conferred homologousprotection against challenge infection with L. interrogans serovarCopenhageni, but did not induce heterologous protection againstinfection with a serovar L. interrogans serovar Manilae (Tables 5 and6). Protection experiments using a different challenge strain, L.kirschneri sorovar Grippotyphosa, indicate that the cross-protectionconferred by the live-attenuated vaccine also protects against adifferent species of Leptospira, other than L. interrogans.

TABLE 5 Protection conferred by immunization with attenuated fcp1−mutant in hamster model of leptospirosis Immunization Challenge No. %Protection (No. Scheme Serovar Animals Died) P-value fcp1− Copenhageni*9 100 (0) <0.001 Heat-killed 9 67 (0) <0.01 PBS 8 — (8) — fcp1−Manilae** 9 100 (0) <0.001 Heat-killed 9 11 (7) NS PBS 8 — (8) — fcp1−Pomona** 8 100 (0) <0.001 PBS 8 — (8) — fcp1− Canicola** 8 100 (0)<0.001 8 — (8) — fcp1− Grippotyphosa** 7 100 (0) 0.02 PBS 7 29 (5) —Representative results of one of four* and one of two** experiments

In contrast, immunization with live fcp1-mutants conferred completeprotection against infection with the five serovars (Tables 5 and 6).Although it doesn't seem to have protection against colonization (Table6), it's important to mention that hamsters are extremely susceptible toleptospirosis where less than 10 leptospires are able to kill theanimals. For that reason, this experimental model may underestimate theefficacy with respect to this endpoint. Nonetheless, these findingsindicate that a single dose of a live attenuated vaccine elicitedcross-protective immunity against serovars belonging to L. interrogans,the species, which encompasses the majority of serovars of human andanimal health importance, but also can confer protection to serovars ofdifferent species among the Leptospira genus (Table 6).

TABLE 6 Immunization with attenuated fcp1⁻ mutant strain protectshamsters against lethal challenge with 10⁸ bacteria via conjunctivalroute. Challenge No. Vaccine Efficacy (%, 95% CI) Vaccine Serovar ExptAnimals Death Death/Colonization fcp1⁻ Copenhageni 4 6, 7, 9, 9 100(90.4-100) 51.6 (34.8-68) Manilae 3 7, 9, 9 100 (88.4-100) 20 (8.4-39.6)Pomona 2 8, 8 100 (82-100) 0 (0-17.1) Canicola 2 7, 8 100 (82.9-100)26.7 (10.5-52.4) Grippotyphosa 2 7, 7 100 (80.3-100) 34 (19.6-58.7)Heat- Copenhageni 2 8, 9 58.9 (36-78.4) 35.3 (17.2-56.4) killed Manilae2 8, 9 5.6 (0-27.7) 0 (0-15.5)

Immunization with Additional Attenuated, Motility-Deficient StrainsConfers Protection Against Leptospirosis Due to Homologous andHeterologous Serovars.

In order to verify that other motility-deficient mutants could bepotential candidates for an attenuated live vaccine, a Himar1 randommutant of L. interrogans serovar Manilae with disruption of gene flaA2was tested. This mutant lacks the expression of FlaA2 and FlaA1 proteinsdescribed also to be part of the sheath of the leptospiral flagellaapparatus. Previous studies showed that this mutant is also avirulent inthe hamster model of infection. Using the same protocol as for the fcp1⁻mutant, a group of 16 animals with the FlaA mutant were vaccinated, andafterward they were challenged for 21 days with the homologous Manilaestrain and the heterologus L1-130 Fiocruz strain.

TABLE 7 Protection conferred by immunization with attenuatedflaA1⁻/flaA2⁻ mutant in hamster model of leptospirosis ImmunizationChallenge No. % Protection Scheme Serovar Animals (No. Died) P-valueflaA1−/flaA2− Manilae 8 100 (0) <0.001 PBS 8 — (8) — flaA1−/flaA2−Copenhageni 8 100 (0) <0.001 PBS 8 — (8) —

The flaA mutant of L. interrogans serovar Manilae (also having adisruption of the flagellar genes, thus lacking of expression of FlaA1and FlaA2) was another potential candidate for live-attenuated vaccine.After vaccination with this strain animals were challenged with ahomologous (L. interrogans serovar Manilae) and heterologous strain (L.interrogans serovar Copenhageni), and similar results were observed aswith the live-attenuated fcp1⁻ vaccine, with complete protection againstdeath after homologous and heterologous challenge (Table 7). All theanimals vaccinated survived the challenge, indicating that the FlaAmutant was able to confer cross-protection against infection.

Immunization with the Attenuated, Motility-Deficient Strains Induce aRobust Antibody Response:

The attenuated vaccine induced a weak agglutinating antibodies (GMT,256; SD, 152.9-429.3) to the homologous serovar, Copenhageni, andundetectable MAT titers against heterologous serovars. In contrats,western-blot experiments with the sera from hamsters after vaccinationwith the live-attenuated fcp1⁻ vaccine showed a strong reaction againsseveral leptospiral proteins for all the different serovars (FIG. 9A),whereas sera from animals vaccinated with the heat-killed bacteriadoesn't seem to produce antibodies against leptospiral proteins (FIG.9B). These results indicate that anti-Leptospira protein antibodies, andnot agglutinating antibodies, are the correlate of vaccine-mediated,cross-protective immunity.

Passive Transfer of Antibodies Generated Against Attenuated, Non-MotileStrains Confers Protection Against Leptospirosis.

To better understand the role of antibodies for the cross-protection,sera from vaccinated hamsters was passive-transferred to naïve animalsbefore lethal challenge. All the control animals, which werepassive-transferred with non-vaccinate sera, died in between days 8 and9 post-infection, whereas all the passively transferred hamsters withthe anti-attenuated vaccine sera survived up to 14 days post-infection(FIG. 10). This result demonstrates that the transfer of antibodiesconfers protection against heterologous infection, indicating thatantibodies against Leptospira protein have a major role on thecross-protection conferred by this vaccine.

In the 1960s, motility-deficient clones describing basic morphologicaldifferences of hooked and straight cells correlated to small colonies insolid media and virulence attenuation in the hamster model, werereported for Leptospira spp. However, at that time it was impossible todetermine the genetic lesion causing the aberrant phenotype, and sincenon-pathogenic strains could also present hook-shaped ends, the authorsconcluded that the results were a coincident simultaneous variation invirulence and visible shape. Bromley et al. (Bromley and Charon, 1979, JBacteriol 137, 1406-1412) characterized a motility-deficient mutant withstraight cell ends and forming uncoiled PFs obtained by chemicalmutation. The authors did suggest that PFs were involved in the shape ofthe cell ends, but they could not rule out the possibility thatmutations were affecting regulatory genes.

The mutant strain described herein appeared via spontaneous mutation,leading to a motility-deficient phenotype, and the identification of thespecific mutation. Mass-spectrometry analyses led to the identificationof a single gene disruption in the motility-deficient clone. Both LV2756 motile and LV 2756 motility-deficient clones had their wholegenomes sequenced (data not shown), confirming the mutation into a geneencoding the Fcp1 protein. The results confirm the relationship betweenthe structure of the PFs, the cell shape, and motility.

Fcp1 is involved in formation of the hook-shaped ends of Leptospiracells, and therefore key for translational motility. Photographicanalysis of wildtype cells during movement showed that the protoplasmiccell cylinder was flexible and bended in response to the gyrations ofthe hooks. Previous results showed that the hook-shaped end ofLeptospira provides counter-torque, but its gyration is not essentialfor propulsion, and that leptospires could have translational motilityonly with the spiral end shape. This observation was supported withmathematical modeling, showing that the minimum energy configuration inthe absence of applied forces or torques is a hook shape, caused by aclockwise rotation of the PFs. It is also known that in translatingleptospires, the gyration of the cells' end caused a spiral-shaped wavesufficient to propel the cells forward, caused by a counterclockwisegyration of the PFs. However, the gyration of the spiral-shaped ends inthe fcp1⁻ mutants, was either too slow or not large enough to yieldsufficient thrust for the bacteria to translate, similar to what happenswith T. phagedenis in low-viscosity media.

Fcp1 is associated with the PFs filament structure in Leptospiraspecies, and essential for the formation of its sheath. Genetic analysisof spirochetal flagellin proteins showed that stiffer PFs can deform thecell cylinder, and larger deformations produced more thrust. The fcp1⁻mutants generated a flagellar structure that when purified lost itsnatural super-coiled form, leading to the inability to change the cellends. Thus, the cells were not able to translate.

A recent MS-based study showed that Fcp1 is the 13^(th) most abundantprotein in the whole proteome of Fiocruz L1-130 WT, and although thisprotein was never described as being involved in the structure offlagella, its abundance is consistent with a structural function. Truebaet al. (Trueba et al., 1992, J Bacteriol 174, 4761-4768) showed that thePFs sheath of Leptospira contains at least one protein with molecularmass of 36 kDa tightly associated with the core.

According to the results, it was found that the FlaA proteins and thecore protein FlaB1 are associated with the purified PFs even in thefcp1⁻ mutants, as shown by MS and western-blot analyses. In vitro and insitu analyses (FIG. 4) showed that the flagella diameter of the fcp1⁻mutants was significantly smaller than that of the wild-type andcomplemented strains.

Furthermore, immuno-EM assays indicated that this protein was associatedwith the surface of the PFs in the Fiocruz L1-130 WT strain (FIG. 6).The same phenotypes were observed in a mutant of fcp1 gene in thesaprophyte L. biflexa serovar Patoc (data not shown). Taken together,these observations corroborate the hypothesis that Fcp1 was essentialfor the formation of the PFs sheath in Leptospira spp., and that thelack of this protein, led to the impaired motility of the cells.

The function of spirochetes' flagellar sheath is still unclear, and thepresence and composition of the sheath differs among species. It isknown that FlaA impacts the shape and the stiffness of PFs and itshelicity in B. hyodysenteriae, but it is present only in the basal bodyregion in B. burgdorferi. A recent study showed that FlaA proteins arenot involved in the formation of the flagellar sheath in Leptospira.Furthermore, the immuno-EM assays did not show expression of FlaA andFlaB1 proteins on the surface of PFs filament with or without Fcp1expression (FIG. 6). However, the mutants that lacked expression of bothFlaA proteins, did express Fcp1 (data not shown), but lacked hook-shapedends and translational motility that rendered straight PFs whenpurified.

Leptospira has four different FlaB proteins, and previous disruption ofFlaB1 gene on L. biflexa serovar Patoc yielded cells with no flagella.Nevertheless, as in other spirochetes, it is not clear the distributionand interaction of these proteins are within the core and sheath of PFs.This indicates that the formation of coiled flagella and hook-shapedends, and their involvement in translational motility, is complex havingmore than one protein involved. The interaction among these proteinsseems to be important for these phenotypes, although it is yet to beunderstood how this interaction works.

It has been shown that rotation of the PF leads to changes in the cellshape caused by resistive forces between PFs and cell body, and thosechanges drive the movement of spirochetes. For that reason, if therewere any perturbation in the flagellar structure itself and/or theinteraction of the flagella with the cell body, it would generate a cellwith impaired motility.

The present data shows that the lack of Fcp1 led to the assembly ofthinner PFs, probably due to the total or partial lack of the sheath.This may cause the loss of its natural tensile strength and theinability to generate enough thrust for translational motility of thecell.

However, mathematical modeling, based on B. burgdorferi motility,recently evaluated the interaction of the PFs with the peptidoglycanlayer (PG) of the bacterial cell body. It was thought that a fluid layermust separate both structures and that motion occurs by resistance thatis generated from fluid drag, instead of friction. Considering that theflagella sheath is responsible for the interaction of the PFs with thePG, it is conceivable that its loss could lead to an impaired adherenceof those two structures, thus causing the inability to slip and engageproperly, eventually resulting in abolishment of proper gyration of thecell end. It is still unclear if the phenotype observed in the presentmutants is due to the event of one of these hypotheses, but a reciprocalaction of both is more likely.

Motility is essential for virulence in Leptospira, and it is directlyinvolved in the dissemination of the agent in different tissues. Theirunique motility behavior may help these organisms to escape from theinnate immune response and rapidly disseminate in mammalian hosts.

A mutant in L. interrogans with disruption of the gene encoding aflagellar motor switch protein (FliY) was previously described showingboth motility and virulence attenuation, but complementation analyseswere not performed. The pathogenesis studies showed that the Fcp1mutants had an attenuated virulence phenotype, where doses as high as10⁸ leptospires with those mutants were unable to kill animals, comparedto a LD₅₀ of 10 leptospires for the wild-type and complemented strains.

Furthermore, it was shown that after infection with the fcp1⁻ mutantsall the animals survived after 30 days of infection with no renalcolonization, even if leptospiral DNA could be detected probably becauseof haematogenous dissemination of non-motile bacteria.

The penetration of the spirochete into the host mucosa is an essentialprocess to establish infection, and motility is, in turn, essential forpenetration. Spirochetes have the ability to cross-epithelial barriers,traverse the intercellular matrix, enter into tissues, disseminate, andfinally cause systemic infections.

By using a conjunctival (CJ) route for infection, which resembles onenatural mode of infection of the agent, the ability of the spirochete tocross the epithelial barrier is critical to reach the bloodstream anddisseminate. Despite of the intraperitoneal (IP) infection, nodissemination of the motility-deficient clone was observed using CJroute, and no deaths occurred, whereas all animals infected with themotile-clone showed dissemination and 100% of death. Nevertheless, theLD₅₀ for CJ route is 5 logs higher than the IP route indicating thatother factors could be acting to prevent the infection like lacrimalfluid, or constant grooming of the animals. However, in vitro studiesshowed that fcp1⁻ mutants were not able to translocate mammalian cellmonolayers after 24 hours, whereas the wild-type and complemented strainhad a translocation rate of 25% and 46%, respectively (FIG. 7). Theseresults indicated that motility was essential for virulence anddissemination and played an important role in the penetration process ofthe spirochete in the host.

Given their unique morphology and structure, spirochetal motility isunusual and by far one of the most complex motility systems amongbacteria. Recent studies showed that the model of the flagellarstructure previously described, indicating that FlaA proteins areresponsible for the PFs' sheath, does not apply for Leptospira. In thestudies, one structural protein of the PFs was identified in leptospiresinvolved in the formation of the hook-shaped end, and in the normalcoiling of flagella.

Also, the data described herein indicates that Fcp1 is essential for theformation of the flagellar sheath, leading to impaired translationalmotility, and furthermore, the inability to penetrate tissues to causeinfection in animal models.

Other Embodiments

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of stimulating an immune response in asubject in need thereof comprising administering a compositioncomprising an effective amount of a motility deficient Leptospirabacteria to the subject, wherein the Leptospira bacteria is deficient inwild-type flagellar-coiling protein 1 (Fcp1).
 2. The method of claim 1,wherein the Leptospira bacteria are live bacteria.
 3. The method ofclaim 1, wherein the Leptospira bacteria are heat-inactivated.
 4. Themethod of claim 1, wherein the Leptospira bacteria have attenuatedbacterial virulence.
 5. The method of claim 1, wherein the compositionfurther comprises an adjuvant.
 6. The method of claim 5, wherein theadjuvant is selected from the group consisting of an oil-in-wateremulsion, a saponin, a cholesterol, a phospholipid, a CpG, apolysaccharide, variants thereof, and a combination thereof.
 7. Themethod of claim 1, wherein administering the composition comprisesproducing antibodies against the motility deficient Leptospira bacteria.8. A method for reducing or treating an infectious disease caused by oneor more Leptospira bacteria in a subject in need thereof comprisingadministering a composition comprising an effective amount of a motilitydeficient Leptospira bacteria to the subject, wherein the Leptospirabacteria is deficient in wild-type flagellar-coiling protein 1 (Fcp1).9. The method of claim 8, wherein the Leptospira bacteria are livebacteria.
 10. The method of claim 8, wherein the Leptospira bacteria areheat-inactivated.
 11. The method of claim 8, wherein the Leptospirabacteria have attenuated bacterial virulence.
 12. The method of claim11, wherein the composition further comprises an adjuvant.
 13. Themethod of claim 12, wherein the adjuvant is selected from the groupconsisting of an oil-in-water emulsion, a saponin, a cholesterol, aphospholipid, a CpG, a polysaccharide, variants thereof, and acombination thereof.